Genetically-encoded technologies that enable the construction of systems that receive, process, and transmit molecular information are essential to advancing basic biological research, applied biomedical research, and biotechnology. RNA switches are a class of ligand-responsive genetic controllers that are being implemented in diverse biological systems to transform our ability to monitor, interface with, and program the dynamic cellular state. While the application of synthetic regulatory RNAs has grown remarkably over the past decade, current approaches to the design of new RNA regulatory elements are inefficient, laborious, and typically do not yield insight into the sequence-structure-function relationships underlying the activities of these molecules in complex biological systems. The goal of the proposed project is to develop new strategies for approaching the measurement, analysis, and design of an important class of RNA switches that incorporate ribozymes as the gene-control element. The goal of the project will be achieved through three specific aims. The first specific aim will focus on developing and validating new data-rich, massively-parallel measurement strategies that leverage next generation sequencing (NGS)-based assays to obtain gene-regulatory and cleavage activity information on millions of RNA switch sequences in a single experiment. The second specific aim will focus on developing new computational methods to perform analyses of large NGS datasets on RNA switch activities to gain and apply new insight into the sequence-structure-function relationships of functional RNA molecules. The third specific aim will apply these new measurement and analysis methods to specific libraries and cellular systems to advance our understanding of the sequence-activity landscape of RNA switches. The successful execution of the project will transform our capacity to rapidly and reliably build these genetic tools for diverse biological systems. In addition, the rich datasets generated through the newly developed methods will be leveraged to uncover new insight into the sequence-activity landscapes underlying this important class of functional RNA molecules and answer long-standing questions in the field. These insights will more broadly advance our understanding of RNA sequence-structure-function relationships and ultimately dramatically improve our capacities for rational design of functional RNA molecules. The assay and analysis methods developed through this project will change the paradigm by which the research community approaches functional RNA design, thereby having a substantially broader impact on the field.